Purified amylase inhibitor and novel process for obtaining the same

ABSTRACT

The present invention provides a purified amylase inhibitor obtained by a superior process that includes extracting ground beans with supercritical carbon dioxide. The invention also provides a method for inducing weight loss in a mammal in need thereof comprising administering to the mammal, an effective amount of an amylase inhibitor obtained by the superior process. A method for improving post-prandial glucose tolerance in a diabetic mammal comprising administering to the mammal, an effective amount of an amylase inhibitor obtained by the superior process, is also provided.

BACKGROUND OF THE INVENTION

Amylase is an enzyme responsible for breaking down the main source ofcarbohydrates in the human diet, namely, starch. The digestion of starchbegins in the mouth where alpha-amylase present in saliva hydrolyzesglucosidic bonds of starch.

By the time thoroughly chewed food reaches the stomach, the averagechain length of starch is reduced from several thousand to less thaneight glucose units. The acid level in the stomach inactivates thesalivary alpha-amylase. Further digestion of starch continues in thesmall intestine by pancreatic alpha-amylase, which is similar to that ofsalivary alpha-amylase.

Decreasing the absorption of carbohydrates by inhibiting the digestionof starch is a very promising strategy in the fields of, for example,weight loss and diabetes mellitus. From a dietary standpoint, it isimportant to target the breakdown of starch since starch is a relativelynonessential nutrient, which provides calories with little benefit.

Amylase inhibitors are derived from various sources, including vegetablealbumins and leguminous plants. Currently, extracts from beans, arebeing utilized most often as a source of amylase inhibitors.

Current methods for purification of amylase inhibitors, which includesconcentrating and drying beans, include the use of heat treatmentsand/or solvents. See U.S. Pat. No. 6,340,699 to Cestaro, et al. However,the use of heat treatments and/or solvents has several drawbacks. Forexample, at high temperatures, certain heat sensitive components of theamylase inhibitor from beans can become degraded. As a result, theamylase inhibitor exhibits a decrease in stability and potency.

In addition, there are environmental and health concerns associated withthe use of solvents during such purification processes. For example,extraction of amylase inhibitors from beans using solvents results inresidual contamination of the extract with the toxic solvent.Furthermore, disposal of the large quantities of solvent required duringpurification processes is a major environmental concern.

Amylase inhibitors that are derived from bean extracts by theconventional heat and solvent methods are not purified, i.e. theycontain impurities and/or contaminants. Examples of such impurities aresolvent residue and inactive components of the beans.

Amylase inhibitors are often added to food products for consumption,such as, for example, powdered drink mixes, prepared shakes, snack bars,etc. The impurities and/or contaminants that remain in the bean extractare associated with negative flavors that render such food productsunappetizing.

Recent studies have indicated that the currently availableamylase-inhibitors work well in vitro, but fail to be effective in vivo.Some of the proffered reasons are that the currently available amylaseinhibitors are unstable in the gastrointestinal tract due to pH, areinsoluble in water, and/or lose potency due to the use of solvents andheat treatments. See Layer, P. et al. Gastroenterology 1985; 88(6):1895-1902.

Therefore, in light of the above deficiencies that exist with currentamylase inhibitors, there is a need for a more pure and potent amylaseinhibitor derived from beans, and a more sophisticated process forobtaining the same.

SUMMARY OF THE INVENTION

These and other objectives have been met by the present invention byproviding an amylase inhibitor obtained by a superior process. Theprocess comprises grinding white kidney beans then extracting the groundbeans under vacuum pressure with supercritical carbon dioxide to removeimpurities, leaving a bean mass. The bean mass is then incubated withdeionized water to obtain a first bean suspension that contains a firstsolid component and a first liquid component. The first solid componentis separated out of the first bean suspension, while retaining the firstliquid component. The first solid component is then incubated withdeionized water to obtain a second bean suspension that contains asecond solid component and a second liquid component. The second solidcomponent is separated out of the second bean suspension, whileretaining the second liquid component. The first and second liquidcomponents are then combined to obtain a final liquid solution. Thefinal liquid solution is subjected to heat exchange to obtain aconcentrated bean extract. The concentrated bean extract is dried and apurified amylase inhibitor is obtained.

The invention also provides a method for inducing weight loss in amammal in need thereof comprising administering to the mammal, aneffective amount of an amylase inhibitor obtained by the superiorprocess.

A method for improving post-prandial glucose tolerance in a diabeticmammal comprising administering to the mammal, an effective amount of anamylase inhibitor obtained by the superior process, is also provided.

DETAILED DESCRIPTION OF THE INVENTION

Applicants have surprisingly discovered that a purified amylaseinhibitor is obtainable by the novel process of the invention.

Amylase inhibitors are glycoproteins that inhibit the enzyme responsiblefor breaking down carbohydrates, namely, amylase. The amylase inhibitorof the invention is derived from beans. Suitable beans for use in theinvention belong to the Phaseolus vulgaris family which includes, forexample, kidney beans. Preferably, the amylase inhibitor is derived fromwhite kidney beans. The amylase inhibitor from white kidney beans issometimes referred to as “phaseolamin.” Preferably, the beans are notgenetically modified beans. The beans are typically small, intact beans.

The amylase inhibitor of the invention is superior to other amylaseinhibitors because it is of a higher degree of purity than amylaseinhibitors obtained by conventional extraction methods, i.e. heat andchemical. Due to the high degree of purity, the amylase inhibitorexhibits improved stability and potency in vitro and in vivo overamylase inhibitors of the prior art.

The amylase inhibitor of the present invention remains stable atelevated temperatures, such as, for example, 120-200° F. Such heatstability allows the amylase inhibitor to be utilized in, for example,food products that are cooked, without losing the beneficial,starch-blocking effects.

The amylase inhibitor also remains intact at extreme pH values. Forexample, the stomach can have a pH of approximately 1-2. The amylaseinhibitor of the invention remains mainly intact under such pHconditions.

In addition, the amylase inhibitor is more potent than the amylaseinhibitors derived from conventional heat/solvent methods. Not beingbound by theory, it is proposed that by avoiding the use of chemicalsolvents, the important tertiary structure of the amylase inhibitor isnot disrupted.

According to the invention, white kidney beans are subject to grindingto produce coarsely ground beans. The beans are ground by any methodknown to those in the art. For example, the beans may be ground bymanual or mechanical means. An example of a manual method for grindingincludes a mortar and pestle. An example of a mechanical method forgrinding includes a grinding mill, such as a Fitzpatrick Millmanufactured by Robinson.

The coarsely ground beans are subject to extraction to remove impuritiesas will be discussed below. The extraction step involves the use ofsupercritical carbon dioxide. Carbon dioxide exists under normalconditions, i.e. ambient temperature and pressure, as a gas. Thecritical temperature (Tc) for CO₂ is 31.06° C. (88° F.) and the criticalpressure (Pc) is 73.8 bar. CO₂ is in a supercritical state when both thetemperature and pressure is higher than its Tc and Pc. In asupercritical state, the CO₂ is essentially a compressed, high densityfluid.

The coarsely ground beans are placed into an extraction vessel (i.e.,extractor) and extracted with supercritical CO₂ under vacuum pressure.Vacuum pressure is typically any pressure which is below atmosphericpressure. In one embodiment, extraction occurs at a temperature of about120° F. to 200° F. for about two hours. Preferably, the extraction stepis performed at a temperature of about 135° F. to 160° F. for about twohours. More preferably, the extraction step is performed at atemperature of about 145° F. for about two hours.

During the extraction step, the supercritical CO₂ fluid passes throughthe ground beans and dissolves and extracts the impurities from thebeans to form a supercritical solution. Thus, the supercritical solutioncontains impurities from the bean. The impurities are typicallynon-polar constituents of the beans and include, for example, lipids,oils, fats, and flavors.

After the extraction has been completed, the supercritical solution isremoved from the extractor via a pressure reduction value. The pressureand the dissolving power of the supercritical fluid is reduced, therebycausing the impurities of the bean to precipitate in a separator. Forpurposes of this invention, the remaining product, substantially free ofimpurities, is referred to as a bean mass. The bean mass contains theglycoproteins, i.e. amylase inhibitors.

The bean mass is then incubated in deionized water to form a first beansuspension. Deionized water is typically water in which ions have beenremoved. The temperature of the deionized water is preferably from about120° F. to about 160° F., more preferably, the deionized water is about140° F. The bean mass is incubated in the deionized water for up toabout 6 hours, more preferably for about 4 hours. During incubation,glycoproteins are extracted from the bean mass.

As mentioned above, after incubation, a first bean suspension isobtained. The first bean suspension contains a first solid component anda first liquid component. The first liquid component contains deionizedwater and glycoproteins (i.e. amylase inhibitor) from the bean mass. Thefirst solid component contains any remaining unextracted componentswhich includes, for example, impurities. The first solid component isthen separated from the first liquid component. The first liquidcomponent is retained in a separate container.

Separation is done by any means known in the art. For example,separation is accomplished by centrifugation or filtration. Filtrationby filter press is preferred. For example, in filtration the first beansuspension is poured over a porous material (e.g., filter), such as afilter paper. The filter allows the passage of the liquid componentthrough the filter and prevents passage of the solid component.

Centrifugation uses centrifugal force to promote solid and liquidseparation. For example, in centrifugation the first bean suspension isplaced in a tube. The tube in then placed in a centrifuge andcentrifugal force is applied. As a result, the solid component gathersin the bottom portion of the tube (i.e. pellet), while the liquidcomponent remains at the top (i.e. supernatant) portion of the tube. Theliquid component is then poured off and retained in a separatecontainer.

Once the first solid component is separated from the first liquidcomponent, the first solid component is incubated, as described above,in deionized water to form a second bean suspension. The second beansuspension contains a second liquid component and a second solidcomponent. The second liquid component contains deionized water andglycoproteins (i.e. amylase inhibitor) from the first solid component.The second solid component contains any remaining unextracted componentswhich includes, for example, impurities. The second solid component isthen separated from the second liquid component by any suitable means,including those discussed above. The second liquid component is retainedin a separate container.

The first liquid component and second liquid component are then combinedto obtain a final liquid solution. The final liquid solution is thensubjected to heat exchange. Heat exchange is a distillation process,which removes water. Heat exchange preferably occurs under vacuumpressure. Apparatus suitable for heat exchange are known in the art.

As a result of the heat exchange step, water is removed from the finalliquid solution to obtain a concentrated bean extract. The concentratedbean extract may contain approximately 25-50% water. More preferably,the concentrated bean extract contains approximately 35% water.

The concentrated bean extract is then dried. Drying of the final beanconcentrate can be accomplished by any suitable means known in the art.For example, in one embodiment, the drying step is performed bylyophilizaton, i.e. freeze drying. The process of freeze drying removesresidual water from the concentrated bean extract by sublimation anddesorbtion.

During lyophilization, the concentrated bean extract is transported in achilled vessel to a freeze dryer for drying. A condenser in the dryingchamber of the freeze dryer traps water removed from the concentratedbean extract, while a vacuum system reduces pressure to facilitate thedrying process. Once the lyophilization process is complete, a purifiedamylase inhibitor is obtained.

In another embodiment, the drying step is performed by utilizing a spraydryer. The spray dryer consists of a feed pump, atomizer, air heater,air dispenser, drying chamber, and systems for exhaust air cleaning andpowder recovery. Air or gas can be used in spray drying. An example of ahot gas which can be used in a spray dryer, includes, but is not limitedto, nitrogen.

The nozzle used in the spray drying process can be, for example, acentrifugal wheel nozzle or a high pressure nozzle. The input (inlet)temperature of the hot air used for spray drying is from about 400° F.to about 500° F., and preferably about 440° F. The output (outlet)temperature of the hot air used for spray drying is from about 150° F.to about 250° F., and preferably about 210° F. In the spray dryingprocess, the concentrated bean extract is sprayed into hot gas, therebyconverting the concentrated bean extract into a free flowing particulatedried bean extract.

After spraying drying, the dried bean extract is rehydrated to obtain arehydrated bean extract. Rehydration is accomplished by the addition ofwater to the dried bean extract. Preferably, the water is deionized. Ina preferred embodiment, approximately 40-70% of the dried bean extractis rehydrated. More preferably approximately 60% of the dried beanextract is rehydrated.

The rehydrated bean extract is then lyophilized, i.e. freeze dried, asdescribed above. The rehydrated bean extract is subjected to freezedrying to obtain the purified amylase inhibitor.

The primary function of amylase inhibitors is to cause temporary, safe,side-effect free malabsorption of dietary starch. Not being bound bytheory, it is believed that the amylase inhibitor of the invention bindsto, and neutralizes, alpha-amylase. By neutralizing alpha-amylase,absorption of carbohydrates is inhibited. As will be discussed below,the amylase inhibitor is effective for inducing weight loss.

As discussed above, alpha-amylase is a naturally occurring starch enzymethat is responsible for the breakdown of starches. For example, inhumans, dietary starches must be broken down into smaller components,such as glucose, in order to be utilized by the body. Starches that areconsumed, but are not broken down into smaller components, such asglucose, are not utilized in vivo. Therefore, by neutralizing the body'salpha amylase, the body's ability to use starches is hindered, andultimately the unused starches are excreted.

Digestion, or the breakdown of starch into glucose, triggers theproduction of insulin. Hence, consuming a starch-rich meal causes anabnormal rise in insulin. Excess insulin triggers hunger and cravings,creating a vicious cycle. One way to end the cycle is to reduce oreliminate the intake of starches. This approach has had very little orno success in inducing weight loss for the long term.

In one embodiment of the invention, a method for inducing weight loss ina mammal in need thereof is provided. The method comprises administeringto the mammal an effective amount of the amylase inhibitor of theinvention.

A mammal in need of weight loss is, for example, any mammal whose weightis detrimental to its health. Another example of a mammal in need ofweight loss is, for example, a mammal that is unhappy with itsappearance due to excess weight. Excess weight of a mammal issubjective. Some examples of mammals in need of weight loss include, butare not limited to, mammals that suffer from diabetes mellitus and/orobesity.

Not being bound by theory, it is believed that the highly pure amylaseinhibitor of the invention induces weight loss by inhibiting theabsorption of starches. In addition, the amylase inhibitor controlscravings associated with carbohydrate absorption. By inhibitingabsorption of dietary starch and controlling cravings associated withcarbohydrate absorption, the amylase inhibitor is effective in inducingweight loss.

The amylase inhibitor of the claimed invention is also used in a mammalsuffering from an impairment of glucose utilization, for example,diabetes mellitus. The impairment in glucose utilization may occur as aresult of a deficiency in the production of insulin by the pancreas, orby ineffectiveness of the insulin produced to utilize glucose. As willbe discussed below, insulin is necessary to the transport of glucosefrom the blood into cells.

Insulin is a hormone naturally produced by the body that is key tocontrolling blood glucose levels. Circulating blood carries glucose thatprovides fuel for the cells. Getting glucose into the cells requiresinsulin, which is produced in the pancreas by beta cells. Normally, thepancreas produces just enough insulin to handle the body's needs. Thisis not the case with hyperglycemia disorders, such as diabetes mellitus(DM), as will be discussed below.

In DM, insulin is either absent, in short supply or unable to performits job efficiently. If glucose cannot get into the cells, itaccumulates in the blood creating increased blood glucose. The amount ofglucose in the blood after consumption of a meal is the postprandialglucose level.

For example, in people who do not have DM, the plasma glucose levelspeaks about one hour after a meal and returns to pre-meal levels withintwo to three hours after a meal. In contrast, those that suffer from DM,the postprandial glucose increases to a higher level and lasts longercompared to those individuals without diabetes. An impairment inpostprandial glucose tolerance can lead to the development of, forexample, cardiovascular disease.

Individuals suffering from DM usually need to ingest insulin to aid inthe absorption of blood glucose into cells. Often, after consuming acarbohydrate rich meal, a diabetic's insulin requirements may markedlyincrease to deal with the high blood glucose levels.

Accordingly, by inhibiting the absorption of dietary starch, the amylaseinhibitor of the present invention will effectively decrease the insulinrequirements of a diabetic mammal. In addition, the amylase inhibitor ofthe present invention will also lower the level of postprandial glucosein the blood, thereby improving postprandial glucose tolerance. Hence,in another embodiment of the invention, a method for improvingpostprandial glucose tolerance in a diabetic mammal is provided.

Preferably, the amylase inhibitor is administered systemically. Systemicadministration can be enteral or parenteral. Enteral administration ispreferred. For example, the amylase inhibitor is easily administeredorally. Liquid or solid (e.g., tablets, gelatin capsules) formulationscan be employed. The formulations can include pharmaceuticallyacceptable excipients, adjuvants, diluents, or carriers.

The amylase inhibitor is also administered in chewable tabletgranulations, with or without sugar, in powdered drink mixes, chewinggum and baking products. In a preferred embodiment, because the amylaseinhibitor is stable under baking temperatures, it is effectivelyadministered in baking mixes such as pancakes, waffles, breads, biscuitsor cookies.

In accordance with the present invention, an effective amount of theamylase inhibitor is any amount known to those skilled in the art toeffectively inhibit the breakdown of dietary starch. Preferably, aneffective amount is administered to a mammal just prior to, during, orshortly after, consuming a starch-rich meal. For example, a typicalpre-meal dosage of the amylase inhibitor is approximately 500 mg to1,500 mg.

In accordance with the invention, mammals include, for example, humans,as well as pet animals such as dogs and cats, laboratory animals such asrats and mice, and farm animals such as horses and cows. Humans are mostpreferred.

EXAMPLE 1 Purification of Amylase Inhibitor by Spray Drying

1. Grinding

Whole, dried, non-genetically modified organism (non-GMO) Phaseolusvulgaris beans were inspected for cleanliness. Upon quality controlapproval of the beans, 1000 g of the dried beans were placed into aFitzpatrick® grinding mill. A #4 screen (course ground size) was used inthe grinding mill. The grinding continued until the beans were theappropriate size.

2. Extraction

The coarsely ground beans were placed into an extraction vessel andextracted with supercritical CO₂. The extraction process occurs undervacuum pressure at about 145° F. for about two hours. The supercriticalCO2 removes the impurities (e.g., lipids, oils, fats, and flavors, etc.)from the coarsely ground beans, leaving a bean mass.

The pressure in the extraction vessel was then reduced. The reductioncauses the impurities from the bean to precipitate from thesupercritical solution and into a separator.

3. Incubation

Deionized water at 140° F. was added to the bean mass and allowed toincubate for 4 hours. During incubation, glycoproteins were extractedfrom the bean mass. A first bean suspension was obtained.

4. Separation

The first bean suspension was then filtered by a filter press toseparate the solid components from the bean suspension. The first liquidcomponent (containing glycoproteins) was retained in a separatecontainer. The first solid component was then incubated in deionizedwater as above to form a second bean suspension. The second beansuspension was then filtered as above to separate the second solidcomponents from the second liquid components. The first and secondliquid components were then combined to obtain a final liquid solution.

5. Heat Exchange

The final liquid solution was subjected to heat exchange to remove waterand to obtain a concentrated bean extract.

6. Drying

The concentrated bean extract was then subjected to spray drying toremove the residual water. A high pressure nozzle was used for the spraydrying procedure. The concentrated bean extract was subjected to hot airwith an input temperature of 440° F. and an output temperature of 210°F., until a dried bean extract was formed.

Approximately 40% (w/w) of the dried bean extract was rehydrated withdeionized water. The rehydrated bean extract was then lyophilized toobtain the purified amylase inhibitor. From the 1000 g of dried beans,approximately 120 g of purified amylase inhibitor was obtained.

EXAMPLE 2 Purification of Amylase Inhibitor by Drying WithLyophilization

1. Grinding

Whole, dried, non-genetically modified organism (non-GMO) Phaseolusvulgaris beans were inspected for cleanliness. Upon quality controlapproval of the beans, 1000 g of the dried beans were placed into aFitzpatrick® grinding mill. A #4 screen (course ground size) was used inthe grinding mill. The grinding continued until the beans were theappropriate size.

2. Extraction

The coarsely ground beans were placed into an extraction vessel andextracted with supercritical CO₂. The extraction process occurs undervacuum pressure at about 14520 F. for about two hours. The supercriticalCO2 removes the impurities (e.g., lipids, oils, fats, and flavors, etc.)from the coarsely ground beans, leaving a bean mass.

The pressure in the extraction vessel was then reduced. The reductioncauses the impurities from the bean to precipitate from thesupercritical solution and into a separator.

3. Incubation

Deionized water at 140° F. was added to the bean mass and allowed toincubate for 4 hours. During incubation, glycoproteins were extractedfrom the bean mass. A first bean suspension was obtained.

4. Separation

The first bean suspension was then filtered by a filter press toseparate the solid components from the bean suspension. The first liquidcomponent (containing glycoproteins) was retained in a separatecontainer. The first solid component was then incubated in deionizedwater as above to form a second bean suspension. The second beansuspension was then filtered as above to separate the second solidcomponents from the second liquid components. The first and secondliquid components were then combined to obtain a final liquid solution.

5. Heat Exchange

The final liquid solution was subjected to heat exchange to remove waterand to obtain a concentrated bean extract.

6. Drying

The concentrated bean extract was then dried by lyophilization to obtainthe purified amylase inhibitor. Approximately 120 g of purified amylaseinhibitor was obtained from the initial 1000 g of beans.

1. A purified amylase inhibitor obtained by a process comprising thesteps of: (i) grinding white kidney beans to produce coarsely groundbeans; (ii) extracting impurities from the coarsely ground beans bysubjecting the beans to supercritical carbon dioxide, under vacuumpressure, to obtain a bean mass; (iii) incubating the bean mass indeionized water to obtain a first bean suspension containing a firstsolid component and first liquid component; (iv) separating out thefirst solid component from the bean suspension and retaining the firstliquid component; (v) incubating the first solid component in deionizedwater to obtain a second bean suspension containing a second solidcomponent and a second liquid component; (vi) separating out the secondsolid component from the second bean suspension and retaining the secondliquid component; (vii) combining the first liquid component and thesecond liquid component to obtain a final liquid solution; (viii)subjecting the final liquid solution to heat exchange to obtain aconcentrated bean extract; (ix) drying the concentrated bean extract;whereby a purified amylase inhibitor is obtained.
 2. The purifiedamylase inhibitor according to claim 1, wherein the separating of steps(iv) and (vi) of the process is carried out by filtering through afilter press.
 3. The purified amylase inhibitor according to claim 1,wherein the separating of steps (iv) and (vi) of the process is carriedout by centrifugation.
 4. The purified amylase inhibitor according toclaim 1, wherein the drying of step (ix) of the process is carried outby spray drying the concentrated bean extract to form a dried beanextract, and wherein the method further comprises the steps of: (x)rehydrating the dried bean extract to form a rehydrated bean extract;and (xi) lyophilizing the rehydrated bean extract.
 5. The purifiedamylase inhibitor according to claim 1, wherein the drying of step (ix)of the process is carried out by lyophilization.
 6. The purified amylaseinhibitor according to claim 1, wherein the extracting of step (ii) ofthe process is carried out at a temperature of about 120-200° F. forabout two hours.
 7. The purified amylase inhibitor according to claim 1,wherein the extracting of step (ii) of the process is carried out at atemperature of about 135-160° F. for about two hours.
 8. The purifiedamylase inhibitor according to claim 1, wherein the extracting of step(ii) of the process is carried out at a temperature of about 145° F. forabout two hours.
 9. The purified amylase inhibitor according to claim 4,wherein about 40-70% of the dried bean extract of step (x) isrehydrated.
 10. The purified amylase inhibitor according to claim 4,wherein about 60% of the dried bean extract of step (x) is rehydrated.11. A method for inducing weight loss in a mammal in need thereofcomprising administering to the mammal, an effective amount of apurified amylase inhibitor according to claim
 1. 12. The methodaccording to claim 11, wherein the mammal is a human.
 13. A method forimproving post-prandial glucose tolerance in a mammal in need thereofcomprising administering to the mammal, an effective amount of apurified amylase inhibitor according to claim
 1. 14. The methodaccording to claim 13, wherein the mammal is a human.